Department of Surgery, Faculty of Medical and Health Sciences, The University of Auckland, Grafton, Auckland, 1023, New Zealand.
Sci Rep. 2022 Jul 20;12(1):12395. doi: 10.1038/s41598-022-16309-w.
Fluorescence in situ hybridisation (FISH) is a powerful molecular technique that enables direct visualisation of specific bacterial species. Few studies have established FISH protocols for tonsil tissue in Carnoy's fixative, accordingly limiting its application to investigate the pathogenesis of tonsillar hyperplasia. Tonsil tissue from 24 children undergoing tonsillectomy for either recurrent tonsillitis or sleep-disordered breathing were obtained during a previous study. The specificity of each of the five FISH probes (Fusobacterium spp., Bacteroides spp., Streptococcus spp., Haemophilus influenzae and Pseudomonas spp.) were successfully optimised using pure and mixed bacterial isolates, and in Carnoy's fixed tonsil tissue. Bacteroides spp. were present in 100% of patients with microcolonies. In comparison, the prevalence of Fusobacterium spp. was 93.8%, Streptococcus spp. 85.7%, H. influenzae 82.35% and Pseudomonas spp. 76.5%. Notable differences in the organisation of bacterial taxa within a single microcolony were also observed. This is the first study to establish a robust FISH protocol identifying multiple aerobic and anaerobic bacteria in Carnoy's fixed tonsil tissue. This protocol provides a strong foundation for combining histological and microbiological analyses of Carnoy's fixed tonsil samples. It may also have important implications on the analysis of microorganisms in other human tissues prepared using the same techniques.
荧光原位杂交（FISH）是一种强大的分子技术，可直接观察特定的细菌物种。很少有研究建立了卡诺氏固定剂扁桃体组织的 FISH 方案，因此限制了其在研究扁桃体增生发病机制中的应用。在先前的一项研究中，对因复发性扁桃体炎或睡眠呼吸障碍而行扁桃体切除术的 24 名儿童的扁桃体组织进行了研究。使用纯菌和混合菌分离株以及卡诺氏固定的扁桃体组织，成功优化了五种 FISH 探针（梭杆菌属、拟杆菌属、链球菌属、流感嗜血杆菌和铜绿假单胞菌）的特异性。100%的患者有微菌落存在拟杆菌属。相比之下，梭杆菌属的患病率为 93.8%，链球菌属为 85.7%，流感嗜血杆菌为 82.35%，铜绿假单胞菌为 76.5%。还观察到单个微菌落中细菌分类群的组织存在显著差异。这是首次建立了在卡诺氏固定的扁桃体组织中识别多种需氧和厌氧菌的稳健 FISH 方案。该方案为结合卡诺氏固定的扁桃体样本的组织学和微生物学分析提供了坚实的基础。它也可能对使用相同技术制备的其他人体组织中微生物的分析具有重要意义。
